The present invention relates to a novel device for harvesting a recombinant protein, which device is characterized by that it comprises a container holding a chromatography media and the container if kept buoyant in nutrient media to maximize the binding of protein in nutrient media with the chromatography media; the device is then removed as harvested protein in a protein-chromatography bound stage. The protein-harvesting device can then be packed in a chromatography column in place of the customary chromatography media and purification performed as desired.
Downstream processing involves cleaning up crude proteins to yield high purity products. Traditionally, these involve use of chromatography columns and highly specialized media to capture and purify the desired proteins. With an exponential rise in the number of protein drugs being developed and marketed, there have been remarkable developments in the field of downstream processing. Still, the time and cost-consuming steps of filtration, chromatography and purification slow down the manufacturing process and add substantial capital cost requirement to establish cGMP-grade manufacturing operations. Recent surveys show that most biopharmaceutical companies consider downstream processing to be their biggest concern since the upstream processing efficiencies have improved creating an imbalance in synchronizing the processes. (7th Annual Report and Survey of Biopharmaceutical Manufacturing. BioPlan Associates, Inc; Rockville, Md.).
The improvements in the downstream processing are mostly focused on creating better chromatography medias such as Protein A or more specific antibodies and converting existing systems into disposable forms, often streamlining the various upstream and downstream processing. Given below is a summary of the art that has been developed or under development:
Single-use downstream chromatography: Novozymes's new patented Dual Affinity Polypeptide technology platform replaces Protein A process steps with similar, but disposable, technology
Stimuli responsive polymers enable complexation and manipulation of proteins and allow for control of polymer and protein complex solubility, which results in the direct capture of the product without centrifuges or Protein A media, from Millipore Corp
Mixed mode sorbents to replace traditional Protein A and ion exchange, for improved selectivity and capacity with shorter residence times. These media, with novel chemistries, include hydrophobic charge induction chromatography, such as MEP, and Q and S HyperCel from Pall Corp
Monoliths, involving chromatography medium as a single-piece homogeneous column, such as Convective Interaction Media monolithic columns from BIA Separations
Simulated moving beds, involving multicolumn countercurrent chromatography, such as BioSMB from Tarpon Biosystems
Protein G (multiple vendors)
Single domain camel-derived (camelid) antibodies to IgG, such as CaptureSelect from BAC
New inorganic ligands, including synthetic dyes, such as Mabsorbent A1P and A2P from Prometic Biosciences
Expanded bed adsorption chromatography systems, such as the Rhobust platform from Upfront Chromatography
Ultra-durable zirconia oxide-bound affinity ligand chromatography media from ZirChrom Separations
Fc-receptor mimetic ligand from Tecnoge
ADSEPT (ADvanced SEParation Technology) from Nysa Membrane Technologies
Membrane affinity purification system from PurePharm Technologies
Custom-designed peptidic ligands for affinity chromatography from Prometic Biosciences, Dyax, and others
Protein A- and G-coated magnetic beads, such as from Invitrogen/Dynal
New affinity purification methods based on expression of proteins or MAbs as fusion proteins with removable portion (tag) having affinity for chromatography media, such as histidine) tags licensed by Roche (Genentech)
Protein A alternatives in development, including reverse micelles (liposomes), liquid-liquid extraction systems, crystallization, immobilized metal affinity chromatography, and novel membrane chromatography systems
Plug-and-play solutions with disposable components (e.g., ReadyToProcess), process development ÄKTA with design of experiments capability, and multicolumn continuous capture, from GE Healthcare.
Affinity Chromatography is a separation technique based upon molecular conformation, which frequently utilizes application specific chromatography medias. These chromatography medias have ligands attached to their surfaces, which are specific for the compounds to be separated. Most frequently, these ligands function in a fashion similar to that of antibody-antigen interactions. This “lock and key” fit between the ligand and its target compound makes it highly specific.
Lectin Chromatography. Many membrane proteins are glycoproteins and can be purified by lectin affinity chromatography. Detergent-solubilized proteins can be allowed to bind to a chromatography chromatography media that has been modified to have a covalently attached lectin.
Immunoaffinity chromatography chromatography media employs the specific binding of an antibody to the target protein to selectively purify the protein. The procedure involves immobilizing an antibody to a column material, which then selectively binds the protein, while everything else flows through.
It is surprising that most of the innovations listed above and those that form prior art involved selective interaction between a target protein and a binding material such as a chromatography media to purify the protein to the limits of pharmacopoeia which currently require a purity greater than 98% (European Pharmacopoeia) and most of manufacturers use an internal control limit of greater than 99.5% purity. However, prior to the commencement of the downstream purification process, the nutrient media needs to be treated to separate the target protein in its crude form as it is not possible to load purification columns with nutrient media without adversely affecting the separation characteristics of these columns and also without excessively prolonging the process of downstream processing that adversely affects the stability of the target protein, besides adding extremely large cost of using large columns, pumps and expensive chromatography media.
There remains a large unmet need to develop a device to capture the target protein non-selectively or selectively and remove it from the nutrient media or a refolding solution prior to subjecting it to customary purification processes. The instant invention is targeted to modify the existing methods of performing protein harvesting or protein capturing prior to purification chromatography to increase the throughput of manufacturing processing without adding expensive and technically challenging modifications.